doi:10.1152/ajpregu.00229.2005 289:1144-1154, 2005. First published Jul 14, 2005;Am J Physiol Regul Integr Comp Physiol

Imbeaud, Valdur Saks and Christophe Pison Vendelin, Jean-Christian Borel, Rachid Hacini, Olivier Chavanon, Sandrine Karen Guerrero, Bernard Wuyam, Paulette Mezin, Isabelle Vivodtzev, Markoexercise training in lung transplant skeletal muscle and mitochondrial creatine kinase is enhanced after Functional coupling of adenine nucleotide translocase

You might find this additional information useful...

45 articles, 24 of which you can access free at: This article cites http://ajpregu.physiology.org/cgi/content/full/289/4/R1144#BIBL

including high-resolution figures, can be found at: Updated information and services http://ajpregu.physiology.org/cgi/content/full/289/4/R1144

can be found at: and Comparative PhysiologyAmerican Journal of Physiology - Regulatory, Integrativeabout Additional material and information

http://www.the-aps.org/publications/ajpregu

This information is current as of September 24, 2005 .  

http://www.the-aps.org/.ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2005 by the American Physiological Society. ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the Americanilluminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization,

publishes original investigations thatThe American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

Functional coupling of adenine nucleotide translocase and mitochondrialcreatine kinase is enhanced after exercise training in lung transplantskeletal muscle

Karen Guerrero,1 Bernard Wuyam,2 Paulette Mezin,3 Isabelle Vivodtzev,2

Marko Vendelin,1 Jean-Christian Borel,2 Rachid Hacini,4 Olivier Chavanon,4

Sandrine Imbeaud,5 Valdur Saks,1 and Christophe Pison1,6

1Laboratoire de Bioenergetique Fondamentale et Appliquee, Institut National de la Sante et de la Recherche Medicale E221,Universite Joseph Fourier, Grenoble, France; 2Laboratoire d’Exploration Fonctionnelle Cardio-Respiratoire, 3Laboratoire dePathologie Cellulaire, and 4Service de Chirurgie Cardiaque, Centre Hospitalier Universitaire de Grenoble, France;5Genexpress, Genomique Fonctionnelle et Biologie des Systemes pour la Sante LGN, UMR 7091, CentreNational de la Recherche Scientifique et Universite Pierre et Marie Curie, Paris, France; 6Departementde Medecine Aigue Specialisee, Centre Hospitalier Universitaire de Grenoble, France

Submitted 1 April 2005; accepted in final form 26 May 2005

Guerrero, Karen, Bernard Wuyam, Paulette Mezin, Isabelle Vi-vodtzev, Marko Vendelin, Jean-Christian Borel, Rachid Hacini, OlivierChavanon, Sandrine Imbeaud, Valdur Saks, Christophe Pison. Func-tional coupling of adenine nucleotide translocase and mitochondrial creatinekinase is enhanced after exercise training in lung transplant skeletal muscle.Am J Physiol Regul Integr Comp Physiol 289: R1144–R1154, 2005;doi:10.1152/ajpregu.00229.2005.—Mechanisms responsible for limitation ofexercise capacity in lung transplant recipients (LR) and benefits gained byexercise training were studied. Mitochondrial respiration parameters, energytransfer, and cell structure were assessed in vastus lateralis biopsies using thepermeabilized fiber technique with histochemical and morphometric mea-surements. Twelve male controls (C) and 12 LR performed exercise trainingover 12 wk. Before exercise training, there were strong correlations betweenexercise capacity (maximal O2 consumption and endurance time at 70%maximal power output) and cellular events, as assessed by percentage of typeI fibers and apparent Km for exogenous ADP. Anticalcineurins were notinvolved in LR exercise limitation, since there were no differences inmaximal mitochondrial rate of respiration before exercise training and noabnormalities in respiratory chain complexes compared with C. Trainingresulted in a significant increase in physiological parameters both at thecellular (apparent Km for exogenous ADP and stimulating effect of creatine)and integrated (maximal O2 consumption, power output at ventilatory thresh-old, maximal power output, and endurance time at 70% maximal poweroutput) levels in LR and C. After the training period, improvements inmaximal O2 consumption and in maximal mitochondrial rate of respirationwere noted, as well as changes in endurance time and percentage of type Ifibers. Because there were no changes in diameters and fiber types, baselinealteration of apparent Km for exogenous ADP and its improvement aftertraining might be related to changes within the intracellular energetic units.After the training period, intracellular energetic units exhibited a highercontrol of mitochondrial respiration by creatine linked to a more efficientfunctional coupling adenine nucleotide translocase-mitochondrial creatinekinase, resulting in better exercise performances in C and LR.

mitochondria; oxidative phosphorylation; creatine/phosphocreatineshuttle; pulmonary transplantation; rehabilitation

LUNG TRANSPLANTATION (LT) has become an established optionfor end-stage respiratory diseases (35). Among such unre-

solved issues as organ shortage and development of progres-sive chronic dysfunction is limitation of maximal O2 consump-tion (VO2 max) after LT, which was been studied since the early1990s without definitive explanations (7, 9, 10, 16, 17, 20, 22,34, 41, 42) (see Ref. 43 for review). VO2 max is broadlydetermined by three major factors: ventilatory capacity, max-imal cardiac output, and O2 consumption by the workingmuscles. Lung transplant recipients (LR) exhibit normal car-diac and ventilatory responses to exercise; therefore, skeletalmuscle dysfunction is thought to explain limitations (7, 9, 10,16, 17, 20, 22, 34, 41, 42, 43). Systemic effects of chronicrespiratory failure before LT (2, 26) and potential effects ofimmunosuppressants, especially the vehicle of cyclosporin A(15), have been proposed as explanations for limitations inVO2 max and decreased exercise capacity after LT. However,the molecular mechanisms of the limitations in O2 consump-tion in this acquired myopathy remain obscure for LR, even ifreductions in skeletal muscle mass and type I fibers play animportant role (2, 9, 26, 41, 43). Energy metabolism is thusinvolved in the genesis of the impairment of exercise capacity.At present, there exists a reliable technique to determine themitochondrial oxidative capacity in human skeletal muscle: thepermeabilized or skinned fiber technique (29, 44). This tech-nique explores mitochondrial oxidative phosphorylation in situwithout isolation of these organelles. This allows the measure-ment of mitochondrial respiration in situ in small biopsysamples. The content of mitochondria in the tissue and theirfunctional capacities can easily be evaluated. In particular, thistechnique has already been used for studies of mitochondrialfunction in inspiratory muscles of patients with chronic ob-structive pulmonary disease (25) and skeletal muscles of hearttransplant recipients before and after rehabilitation (46).

The aims of the study were to look for alterations in theintracellular organization in energy metabolism, i.e., mitochon-drial respiration and energy transfer via phosphocreatine shut-tle and, if present, the relevance of mitochondrial bioenergeticsin limitations of skeletal muscle performance in LR (3) and

Address for reprint requests and other correspondence: V. A. Saks, Labo-ratoire de Bioenergetique Fondamentale et Appliquee, Institut National de laSante et de la Recherche Medicale E221, Universite Joseph Fourier, 2280 ruede la Piscine, BP53X 38041, Grenoble Cedex 9, France (e-mail: e-mail:Valdur.Saks@ujf-grenoble.fr).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 289: R1144–R1154, 2005;doi:10.1152/ajpregu.00229.2005.

0363-6119/05 $8.00 Copyright © 2005 the American Physiological Society http://www.ajpregu.orgR1144

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

whether home exercise training can improve these abnormali-ties (4, 34).

METHODS

Subjects and Experimental Design

Twelve healthy males, recruited from acquaintances of investigatorsand free of any medications, were enrolled in the study as a control group(C). Twelve LR (35 � 28 mo post-LT) were also included. Antirejectionmaintenance treatment consisted of prednisone, 0.1 mg/kg on average,tacrolimus, and mycophenolate mofetil (Table 1). Most LR receivedantihypertensive treatments such as calcium blockers or angiotensin-converting enzyme inhibitors. LR were free of acute and/or significantchronic rejection (except the 49-yr-old LR) or infection during the study.C and LR were examined before and after a 3-mo period of exercisetraining. Participants gave their voluntary consent. The protocol wasapproved by the ethics committee of our institution.

Body Composition, Pulmonary Function, and Exercise Testing

Fat-free mass was measured with bioelectrical impedance analysisat 50 kHz, and the equation from Kyle et al. (15) was used. Pulmonaryfunction was measured using standard techniques according to Amer-ican Thoracic Society/European Respiratory Society recommenda-tions (23). Lung function measurements consisted of 1) forced expi-ration from which 1-s forced expiratory volume, forced vital capacity,and expiratory flow-volume loops were derived and 2) thoracic gasvolumes, which were determined in a plethysmograph (MedicalGraphics, St. Paul, MN). Pulmonary function measurements areshown in Tables 1 and 2.

Maximal incremental and constant workload cycle exercise wasperformed as detailed below.

First, for maximal incremental cycling exercise, subjects wereseated on an electrically braked ergocycle (Ergometer 900PC, Ergo-line) driven by Vmax system 229 software (SensorMedics, YorbaLinda, CA). Gas exchanges were analyzed by the standard open-circuit method with an automated computer analysis system (Vmax2130 automated spirometer, SensorMedics). Continuous monitoringof gas exchange provided measurements of O2 uptake (VO2), CO2

excretion, and minute ventilation (VE). The expired O2 and CO2

fractions were analyzed breath by breath with zirconium electrochem-ical and infrared cell gas analyzers, respectively. Expiratory flow wasmeasured with a hot wire pneumotachograph held with a mouthpiece.After 5 min of resting and 2 min of warm-up at 20 W, a progressivestepwise exercise was performed up to the individual’s maximal capacity.Workload increments were 5 and 15 W/min for LR and C, respectively.Subjects were considered to have achieved maximal VO2 if at least threeof the four following criteria were met: 1) no further increase in VO2 withincreasing workload, 2) respiratory exchange ratio �1.1, 3) maximalheart rate �90% of the maximal predicted value, and 4) plasma lactateconcentration �8 mmol/l. In other cases, tests were considered assymptom-limited exercise testing. Power output at the ventilatory thresh-old (Wventilatory threshold) was determined as the power output for which anabrupt and nonlinear increase in VE (minimal value of VE/VO2) wasobserved over the incremental test.

Second, for endurance cycling exercise, subjects were seated on theergocycle, and gas exchange was analyzed under the same conditionsas previously described. After 5 min of resting and 2 min of warm-upat 20 W, a steady-state exercise was performed up to exhaustion.Exercise intensity was set at 70% of the maximal power output (W)reached by the subjects during their initial maximal incrementalexercise. After exercise training, the maximal exercise test was re-peated. The endurance test was also repeated on the same day, withthe same load as the initial test, and the patient was allowed to

Table 1. Clinical characteristics of LR and C before exercise training

Age, yrPreoperative

DiagnosisType of

ProcedureMonthsPost-LT FEV1, liters FEV1/FVC VE/MVM VE/VO2 max VO2 max l/min Wmax, W

Max HR,% predicted

20 Cystic fibrosis Double 22 4.20 0.84 0.55 36.6 2.42 165 9037 Cystic fibrosis Double 5 3.59 0.96 0.52 42.5 2.03 100 7938 Emphysema Double 60 3.72 0.87 0.54 48.2 2.20 120 7740 Cystic fibrosis Double 25 2.77 0.74 0.56 37.0 2.14 150 9245 Emphysema Double 59 2.16 0.79 0.63 43.2 1.49 55 6947 Emphysema Double 5 2.04 0.63 0.68 41.4 2.03 60 7347 Histiocytosis Double 10 3.53 0.88 0.47 47.0 2.30 105 9549 Cystic fibrosis Double 82 0.94 0.37 0.48 63.2 2.24 34 6857 COPD HL 76 2.27 0.77 0.57 40.7 1.97 80 6658 PAH Double 2 1.90 0.95 0.63 55.6 1.16 40 7060 IPF Single 51 1.63 0.70 0.46 46.8 2.16 70 7570 Emphysema Single 36 1.36 0.70 0.72 39.1 1.80 50 63

47�13 35�28 2.51�1.04* 0.77�0.16 0.64�0.23 46.2�8.6* 1.25�0.56* 85�43* 76�11*22 C 4.87 0.86 0.58 29.2 2.89 285 9422 C 4.67 0.85 0.44 31.1 3.11 225 8526 C 4.53 0.90 0.62 36.2 2.46 210 9930 C 5.45 0.81 0.68 28.8 3.16 315 9930 C 4.71 0.82 0.45 27.2 3.22 315 9932 C 3.79 0.83 0.53 36.6 2.90 180 9135 C 5.00 0.84 0.56 31.4 3.43 165 8544 C 4.55 0.89 1.29 35.0 2.74 180 10144 C 3.14 0.79 0.59 32.1 2.51 165 10550 C 4.83 0.78 0.62 37.2 2.76 253 8854 C 3.91 0.76 0.79 29.5 2.08 180 9655 C 4.07 0.75 0.53 40.9 2.84 210 81

37�12 4.46�0.63 0.82�0.05 0.57�0.08 32.9�4.1 2.88�0.67 225�56 94�7

Values in bold are means � SD. COPD, chronic obstructive pulmonary disease; PAH, pulmonary arterial hypertension; IPF, interstitial pulmonary fibrosis;HL, heart lung transplant; LT, lung transplant; FVC, forced vital capacity; VE, external ventilation volume; MVM, maximal volume exchanged per minute [37 �1-s forced expiratory volume (FEV1)]; VO2 max � maximal O2 consumption; Wmax � maximal power output; Max HR, maximal heart rate. *P � 0.0002 forcomparisons between lung transplant recipients (LR) and controls (C) before exercise training.

R1145MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

continue to exercise to the limits of tolerance (to evaluate anypotential improvement in exercise endurance).

Exercise Training Program

LR and C underwent exercise sessions, three times per week, for 12wk at home on a cycle ergometer (Ergometer 900PC, Ergoline). Theywere directly supervised for the first sessions by a physiotherapist athome and via phone each subsequent week. For security reasons andcontrol purposes, subjects had a cardiofrequency meter (S610i, Polar,Kempele, Finland) with memory. Every month, these monitors weredownloaded to a personal computer to check that planned exercisesessions were achieved. Exercise consisted of cycling at 50% andprogressively increasing to 80% of previously determined Wmax for10 min followed by 5 min at 30% Wmax, three times per session in Cand LR.

Skeletal Muscle Biopsy and Muscles Studies

Local anesthesia (1% xylocaine) was injected into the skin of thethigh without crossing the muscle aponeurosis to avoid mitochondrialuncoupling. One part of the biopsy sample was immediately immersedin ice-cold relaxing medium (solution A complemented with 0.2%BSA) and used for preparation of fibers (see Preparation of perme-abilized muscle fibers). The second part was immediately frozen inisopentane, which was cooled by liquid nitrogen for enzymatic,morphological studies. The third part of the biopsy sample was fixedwith 2.5% glutaraldehyde for electron microscopic study.

Electron microscopy. Semifine cross sections (1–2 �m) were preparedwith a Leica Microsystems microtome and stained with toluidine blue. Finesections (0.07 �m) were prepared with a Reichert-Jung microtome, stainedwith uranyl acetate and lead citrate, mounted on 300-mesh grids, andexamined under a CM10 Philips electron microscope.

Muscle fiber types. The muscle cross sections (10 �m) werepreincubated at pH 4.35. After they were stained for myosin ATPase,fibers were visually counted to determine the percentage and the meandiameter of each fiber type (8). Morphometric measurements wereperformed with Histo Biocom software (Les Ullis, France).

Preparation of permeabilized muscle fibers. The nonfrozen parts ofthe biopsy samples that had been immediately immersed in ice-coldrelaxing medium (solution A complemented with 0.2% BSA) wereused in these experiments. Thin bundles of fibers were then separatedby sharpened needles in ice-cold solution A with 0.2% BSA. Afterdissection, fibers were incubated in solution A with 50 �g/ml saponin,30 min, at 4°C and mild stirring. They were then washed three timesin solution B with 0.2% BSA, 10 min, at 4°C and mild stirring toeliminate the detergent and the endogenous metabolites (29).

Determination of the rate of mitochondrial respiration inskinned fibers. The main mitochondrial reactions studied to characterizethe muscle energy metabolism are schematically shown in Fig. 1. The

Fig. 1. Representation of the different mitochondrial compartments (matrixand intermembrane space) delimited by the outer and the inner membranes.Mitochondrial respiratory chain (localized in the inner membrane) as well asits substrates (�) and inhibitors (�) consume O2 and create the proton gradientnecessary for the synthesis of ATP. The functional coupling of adeninenucleotide translocase (ANT)-mitochondrial creatine kinase (MtCK) is alsorepresented to show the control of respiration by creatine (Cr) in the case ofoxidative muscle by the regeneration of local ADP in the intermembrane space.On the outer mitochondrial membrane, porin allows ADP and Cr to go insidemitochondria. PCr, phosphocreatine; TMPD, tetramethylpentadecane; VDAC,voltage-dependant anion channel; F0F1, mitochondrial ATPase synthase.

Table 2. Effects of exercise training

Exercise TrainingBefore

vs. Aftertraining

P C vs. LR PBefore After

BMI, kg/m2

C 24�3.4 24�2.7 0.58 0.05LR 22�2.7 22�2.0 0.25

FEV1, %predictedC 108�12 106�9.7 0.20 0.0003LR 74�24 77�26 0.44

FVC, %predictedC 109�13 109�11 0.82 �0.0001LR 78�16 82�18 0.19

Wmax, WC 224�56 255�67 �0.001 �0.0001*LR 85�43 98�42 0.04

Pventilatory threshold, WC 112�36 125�42 0.09 �0.0001LR 42�21 52�25 0.02

VO2max, l/minC 2.9�0.67 3.1�0.70 0.01 �0.0001LR 1.2�0.56 1.4�0.51 0.06

VO2max, %predictedC 101�16 113�23 0.02 �0.0001LR 63�20 68�19 0.07

Endurance time at 70%Wmax, min

C 36�19 41�13 0.08 0.04LR 20�10 31�14 0.01

Fiber type I, %C, n � 11 42�21 36�8.6 0.37 0.0008LR, n � 11 16�6.1 24�17 0.14

V0, nmol O2�min�1�mg dry wt�1

C, n � 11 0.51�0.34 0.75�0.52 0.10 0.51LR 0.68�0.36 0.79�0.56 0.57

Vmax, nmol O2�min�1�mg dry wt�1

C, n � 11 3.5�1.5 4.3�2.9 0.38 0.50LR 3.2�1.7 2.9�1.3 0.56

VAtr, nmol O2�min�1�mg dry wt�1

C, n � 10 0.71�0.38 1.5�1.0 0.04 0.49LR 0.79�0.35 1.1�0.68 0.12

Values are means � SD. BMI, body mass index; Vo, basal mitochondrialrespiration in the presence of glutamate (5 mM) and malate (2 mM); Vmax,maximal mitochondrial respiration rate; Vatr, mitochondrial respiration rate inthe presence of atractyloside, an inhibitor of adenine nucleotide translocase(ANT). ANOVA with 2 factors: I) effects of exercise training and 2) groupeffect. *Significant interaction between the effects of exercise training and thegroups: LR vs. C.

R1146 MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

rates of VO2 were recorded by using a two-channel, high-resolutionrespirometer (Oroboros Oxygraph, Paar, Graz, Austria) in solution B(measurement of ADP kinetics of respiration) or using KCl solution forcytochrome c test (for composition, see below). Activity of respiratorychain complexes was determined in KCl solution with 2 mg/ml BSA plus5 mM glutamate, 2 mM malate, 2.5 �M rotenone, 5 mM succinate, 32�M myxothiazol, 5 mM ascorbate, 1 mM tetramethylpentadecane, and 5mM KCN. Determinations were carried out at 25°C; solubility of O2 wastaken as 215 nmol/ml (14).

Solution A contained (in mM) 1.9 CaK2EGTA, 8.1 K2EGTA (freecalcium concentration of 0.1 �M), 9.5 MgCl2, 0.5 DTT, 50 potassium-MES, 20 imidazole, 20 taurine, 2.5 Na2ATP, and 15 phosphocreatine, pH7.1, adjusted at 25°C. Solution B contained (in mM) 1.9 CaK2EGTA, 8.1K2EGTA, 4.0 MgCl2, 0.5 DTT, 100 potassium-MES, 20 imidazole, 20taurine, and 3 K2HPO4, pH 7.1, adjusted at 25°C.

KCl solution for cytochrome c test contained (in mM) 125 KCl, 20HEPES, 3 magnesium acetate, 5 KH2PO4, 0.4 EGTA, and 0.3 DTT,pH 7.1, adjusted at 25°C.

For respiration rate determinations, 2 mg/ml BSA was added; 5mM glutamate and 2 mM malate were added as substrates.

Reagents. All reagents were purchased from Sigma except ADP,which was obtained from Boehringer-Mannheim.

Statistical Analysis

Values are expressed as means � SD. Comparisons were madeusing two-way ANOVA, with one factor corresponding to compari-sons between C and LR and the second comparing before and afterexercise training. Significant interactions within these two factors arereported. P � 0.05 was taken as the level of significance.

RESULTS

Comparisons Between C and LR Before Exercise Training

Table 1 reports clinical characteristics in LR and C. The male-to-female ratio was 12:0 for C and 10:2 for LR. Body-mass index waslower, as well as 1-s forced expiratory volume and forced vitalcapacity in LR (Table 2). No subject exhibited ventilatory limits

during maximal cycling exercise test. As expected, Wmax, Wventilatory

threshold, VO2 max (in l/min or %predicted), and endurance times weremuch lower in LR compared with C (Tables 1 and 2).

Histochemical determination of fiber types is reported in Fig.2A for C and Fig. 2C for LR. LR had a significantly lowerpercentage of type I fibers compared with C (Table 2). Figure 3,A and C for C and LR, respectively, shows examples of mito-chondrial arrangement in the fibers along the direction of sarco-mere orientation analyzed by electron microscopy. Most mito-chondria were localized close to the z-line, as characteristic for theglycolytic and oxidative-glycolytic fibers. In some places, a smallamount of intermyofibrillar mitochondria was seen.

Exercise Training Effects in C and LR

Exercise training had physiological effects in both groupswith regard to Wmax, Wventilatory threshold, and VO2 max (Table 2),with a trend toward more pronounced effects in LR. Endurancetime increased by 55% in LR, whereas it did not reachsignificance in C. External ventilation significantly decreasedafter exercise training for comparable workloads in C and LR(data not shown). Interestingly, the fiber-type distribution (Fig.3B for C and Fig. 3D for LR) and diameters of both fiber typeswere not significantly changed by exercise training. Analysisof multiple electron micrographs did not reveal any visiblechanges in the arrangement of mitochondria in the cells afterexercise training (Fig. 3B in C and Fig. 3D in LR).

Mitochondrial Respiration Before and After the ExerciseTraining Period

Before exercise training, LR mitochondrial respiratorychain capacity was similar to C. The basal rate of respirationwas obtained in the presence of 5 mM glutamate and 2 mMmalate, substrates of the complex I of the respiratory chain(Figs. 1 and 4). The usually low basal rate of respiration

Fig. 2. Identification of fiber type accordingto ATPase reactivity. Type I fibers appeardarker, whereas type II fibers appearbrighter. A and B: fibers from controls (C)before and after rehabilitation, respectively.C and D: fibers from lung transplant recipi-ents (LR) before and after exercise training,respectively. Note that LR have a higherproportion of type II fibers compared with Cand that the proportion of each type of fiberdoes not change with training in both groups.Bars � 60 �m.

R1147MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

corresponds to the state 2 of the respiratory chain where themembrane potential (gradient of electrochemical potential ofprotons) is generated. After addition of ADP, transported intomitochondrial matrix in exchange for ATP by adenine nucle-otide translocase (ANT) (Fig. 1), the membrane potential isused for the synthesis of ATP (state 3 of respiratory chain).Due to a decrease of the membrane potential, the respirationrate in state 3 is increased manyfold in the presence of ADP ata high concentration (2 mM) to achieve the maximal rate ofrespiration (Vmax; Fig. 4D). This phenomenon is called accep-tor control, and the ratio of the respiration rate in state 3 to thatin state 2 is called the acceptor control ratio (ACR). Figure 4Dshows that basal respiratory rate was very low and ACRexceeded 8, giving evidence for an intact inner membrane andeffective coupling between oxidation and phosphorylation inthe skeletal muscle mitochondria in LR and C. The integrity ofthe outer mitochondrial membrane was assessed by the cyto-chrome c test (14). The absence of the effect of exogenouscytochrome c (8 �M) on the respiration rate in the KClmedium showed integrity of this membrane (Fig. 4D). Addi-tion of atractyloside resulted in a decrease of respiration tostate 2 (Table 2, Fig. 4, A and B). In C, basal respiratory rateand Vmax values were close to those found in the literature (25,46), and the ACR values indicated a high degree of couplingbetween oxidation and phosphorylation (data not shown). Withglutamate and malate as substrates, the basal respiratory rateand Vmax values were not significantly different in C and LR(Table 2). Activities of respiratory chain complexes (by acti-vating and inhibiting fragments of the respiratory chain) didnot reveal any alteration of the oxidative phosphorylation in themitochondrial inner membrane of LR before and after exercisetraining compared with C (data not shown).

Effect of ADP and creatine on mitochondrial respirationbefore exercise training. Successive additions of exogenousADP in increasing concentrations up to 2 mM resulted in anincrease of the respiration rate. For high concentrations of ADP(2 mM), Vmax was reached (Fig. 4, A and B). Apparent Km forexogenous ADP was calculated from the double-reciprocal plotof these kinetic data (Fig. 4C). Apparent Km for exogenousADP represents the sensitivity of mitochondria for ADP, in-cluding on the one hand the permeability of the outer mito-chondrial membrane (voltage-dependent anion channel) and onthe other hand cytoplasmic diffusion restrictions for exogenousADP induced by cellular structures (31, 36). When theseexperiments were repeated in the presence of 20 mM creatine,the kinetics of respiration regulation by exogenous ADP waschanged, i.e., addition of small amount of ADP rapidly in-creased the respiration rate (Fig. 4, B and C) by decreasing theapparent Km for exogenous ADP (increasing affinity for ADP).This is explained by the functional coupling of the mitochon-drial creatine kinase (MtCK) with ANT (Fig. 1). The Vmax ofrespiration in the presence of creatine was the same or higherthan in the absence of creatine (Fig. 4B). The functionalcoupling between MtCK and ANT is the natural mechanism ofregulation of respiration by ADP locally produced from mito-chondrial ATP by the MtCK localized at the outer surface ofthe inner mitochondrial membrane (Figs. 1 and 7). Dramaticdifferences were found in the apparent Km values for exoge-nous ADP in regulation of respiration between C and LRbefore exercise training (Fig. 5). In C, apparent Km for exog-enous ADP (131 � 26 �M) was characteristic of skeletalmuscle having mixed fiber composition, with intermediatevalues of apparent Km for ADP oxidative (250–300 �M) andglycolytic (10–20 �M) fibers. This average value of apparent

Fig. 3. Electron microscope images. A andB: regular organization of pairs of mitochon-dria at the z-line level in a control quadricepsmuscle before and after training, respec-tively. C and D: samples were taken fromLR quadriceps muscle before and after train-ing. In this case, mitochondrial regular ar-rangement is conserved on both sides of thesarcomeres. Note that mitochondrial mor-phology is normal in all images. Bars � 1�m.

R1148 MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

Km corresponded to the muscle fiber-type distribution in controlfibers (Table 2). In the presence of creatine (20 mM), the apparentKm for ADP decreased significantly to 57 � 31 �M because ofthe local regeneration of ADP by MtCK in the intermembranespace. This ADP was more efficient in stimulation of respirationthan the exogenous ADP. This explains the drop in Km and showsinteraction between MtCK and ANT in skeletal muscle mitochon-dria. The creatine effect on the apparent Km for exogenous ADPwas calculated as the difference between Km in the absence ofcreatine and in the presence of creatine.

In LR before exercise training, although there was no statisti-cally significant difference with C regarding the respiration rates(basal respiration rate and Vmax), the apparent Km was signifi-cantly lower (94 � 34 �M) compared with C. This low value ofapparent Km was accompanied by a decay of type I oxidativefibers in LR fiber (Table 2). Moreover, the stimulating effect ofcreatine was less apparent in LR (Fig. 5). The partial loss of thecreatine effect resulted from the glycolytic metabolism, whichdoes not rely on coupled energy transfer systems (25).

Effects of ADP and creatine on mitochondrial respirationafter exercise training. Figure 5 shows the dramatic effects ofphysical training on apparent Km for exogenous ADP in both Cand LR. In C, apparent Km for exogenous ADP increased from131 � 26 to 260 � 67 �M, whereas in the presence of creatinethis parameter remained very low (Fig. 5). These data under-line the increased efficiency of the respiration regulation bycreatine in C. The same pattern is seen in LR: the apparent Km

increased from 94 � 34 to 203 � 62 �M and the creatine effectbecame highly significant after exercise training (Fig. 5). Vmax

values were unchanged after exercise training in all conditions,but Vmax values with creatine increased by 58% in LR (P �0.05) (data not shown).

Correlations of VO2 max and Endurance Time at 70% Wmax

Before and After Exercise Training

Before exercise training in C and LR (n � 24), VO2 max

(ml �kg�1 �min�1) was correlated with percentage of type I

Fig. 4. Examples of O2 consumption (VO2) by permeabilized quadriceps fibers. Top traces (A, B, D): changes in O2 concentration ([O2]) vs. time. Bottom traces(A, B, D): rate of respiration (derivative function of VO2). Decrease in respiration after substrate addition is artifactual. A: kinetics of regulation of respirationby exogenous ADP in solution B. This experimental trace shows the dependence of respiration on concentration of ADP. Mitochondrial respiration increases withincreasing concentrations of ADP in a range from 0.05 to 2 mM to reach the maximal rate of respiration (Vmax). From this ADP-stimulated respiration, aMichaelis-Menten representation can be obtained as mitochondrial respiration rate is a function of ADP concentration (see Fig. 2C). At the end of the measure,cytochrome c (Cyt c) addition does not change the respiration, indicating that the outer membrane is intact. Moreover, atractyloside (Atr) addition results in adecrease of respiration back to the basal rate of respiration (V0), showing that we observe mitochondrial respiration. B: kinetics of the regulation of respirationby exogenous ADP in the presence of Cr in solution B. In the presence of Cr, Vmax is reached at lower concentrations of ADP. This is explained by the controlof Cr on oxidative phosphorylation. At the end of the measure, Cyt c addition results in no damage of the outer membrane. Moreover, Atr addition results ina decrease in respiration back to V0. C: double reciprocal plot of Michaelis-Menten graph: Lineweaver-Burk representation. This graph allows determination ofthe apparent affinity of mitochondria (apparent Km) for ADP. In the absence of Cr, apparent Km value is rather high (more than 100 �M), meaning a low affinityof mitochondria for ADP. However, in the presence of Cr, apparent Km is low, indicating a higher affinity of mitochondria for ADP. Vn, normalized rate ofrespiration. D: test of integrity of the outer mitochondrial membrane in KCl medium. V0 of fibers is obtained in the presence of glutamate (Glu; 5 mM) and malate(Mal; 2 mM). Vmax is reached by the addition of 2 mM ADP. Cyt c was then added (8 �M). There is no change in VO2, thus demonstrating that the outermitochondrial membrane is not altered; otherwise, there would have been an increase in the rate of respiration. dw, Dry weight.

R1149MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

fibers (r � 0.69, P � 0.001), apparent Km for exogenous ADP(r � 0.64, P � 0.001), and Vmax (r � 0.44, P � 0.03) (Fig. 6).Wmax was correlated with percentage of type I fibers (r � 0.77,P � 0.0001) and the apparent Km for exogenous ADP (r �0.58, P � 0.004). Endurance time at 70% Wmax was related topercentage of type I fibers (r � 0.72, P � 0.0001), fat-freemass (r � 0.53, P � 0.007), and the apparent Km for exoge-nous ADP (r � 0.48, P � 0.02).

After exercise training, VO2 max (%predicted) changes wererelated to Vmax changes in C and LR (r � 0.55, P � 0.007).

DISCUSSION

Using the saponin-skinned fiber technique, we provided newinsights in the mechanisms of limitation of exercise capacityand the benefits of exercise training in LR in relation to C.Before exercise training, LR mitochondrial respiratory ratesdid not reveal any alterations compared with C rates, excludingdeleterious effects of anticalcineurins. Before exercise training,there were strong correlations between exercise capacity(VO2 max and endurance time) and cellular events, as assessedby the percentage of type I fibers (apparent Km for exogenousADP). Exercise training resulted in significant improvementsin bioenergetics at both the cellular (apparent Km for exoge-nous ADP and stimulating effect of creatine) and the integratedlevel (VO2 max, Wmax, endurance time), which were initiallylower in LR compared with C. Changes in VO2 max and Vmax

were related. Changes in endurance time and percentage oftype I fibers were also correlated. Because there were nochanges in diameters and fiber types, baseline alteration ofapparent Km for exogenous ADP and its improvement aftertraining might be related to changes within the intracellularenergetic units (ICEUs) (Fig. 7). After training, as shown byICEUs, there was a higher control of mitochondrial respirationby creatine linked to a more efficient functional coupling ofANT-MtCK, resulting in better exercise performances in Cand LR.

Comparisons Before Exercise Training Between LR and C

Before exercise training, LR achieved lower VO2 max, Wmax,and endurance time compared with C (Table 2) as previouslyreported by Williams et al. (42, 43) and others (7, 9, 10, 16, 17,20, 22, 34, 41). Pre- and posttransplant factors may explainimpairment in exercise capacity in LR. Pretransplant factorsinclude long-standing deconditioning, poor nutritional status,direct and indirect effects of chronic hypoxia, and corticoste-roid exposure (see Refs. 2 and 3 for reviews) and mainly resultin a reduction of type I fibers in skeletal muscles (11, 26).End-stage respiratory diseases via a systemic inflammation andhypoxia (2, 3) lead to an inhibition of anabolic transcription

Fig. 5. Comparison of ADP sensitivity of mitochondrial respiration of C andLR skeletal muscle before and after exercise training with and without Cr. Inthe absence of Cr, apparent Km for ADP in C is characteristic for mixedskeletal muscle (open bars). That of the LR group, however, is significantlylower (gray bars), closer to that of glycolytic fibers. In the presence of Cr,apparent Km for ADP decreases in C as well as in LR. After rehabilitation(hatched bars), in the absence of Cr, apparent Km for ADP increases 2-fold inC and LR. In the presence of Cr, apparent Km for ADP decreases in C and LR.Values are means � SD. *P � 0.001 before vs. after exercise training for Km

for ADP without Cr. Right: Km for ADP with Cr. Differences between Km forADP with and without Cr are highly significant (P � 0.0005 in C, P � 0.0009in LR), whereas these difference are more pronounced in C vs. LR (P � 0.02).

Fig. 6. Correlations for C (■ ) and LR (E) before exercise training (n � 24)between maximal O2 consumption (VO2 max) and bioenergetics parameters. A:percentage of type I fibers. B: apparent Km for exogenous ADP. C: mitochon-drial Vmax.

R1150 MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

factor, such as myoD, with a resulting preferential ubiquinationof the myosin heavy chain, as in cancer cachexia (1). Amongposttransplant factors, cardiac and ventilatory limitations donot play a significant role because, in LT, the heart is notdenervated and ventilatory limits were not reached in ourpatients, despite lower pulmonary function tests (Tables 1 and2). In rats, it has been shown that anticalcineurins via thevehicle of cyclosporin A are responsible for reduced exerciseendurance time by a decay in mitochondrial oxidative phos-phorylation via reduced complex I and IV activities (28). In aclinical setting such as LT, after cardiac transplantation, im-munosuppressive therapy effects are negligible on mitochon-drial respiration (46) since Vmax in LR was not different fromC (Table 2). Moreover, LR patients received tacrolimus asanticalcineurin therapy and were not exposed to cyclosporin A(15). It appears that skeletal muscle dysfunction acquiredbefore LT plays a central role in exercise limitation after LT, asshown by Wang et al. (41) in LR and Richardson et al. (26) in

chronic obstructive pulmonary disease. Our results confirmedthat percentage of type I fibers plays a major role in VO2 max,Wmax, and endurance time in LR and C (26, 41) (Table 2 andFigs. 2 and 6).

Before exercise training, besides the important role of type Ifibers, analyses of bioenergetic parameters such as apparent Km

for exogenous ADP, Vmax, and stimulating effect of creatine onrespiration give new insights into cell organization related toATP production and channeling of energy transfer after thetraining period (Figs. 1, 6, and 7). Before exercise training inLR, our data showed that mitochondrial rates of respiration(Vmax, basal rate, and mitochondrial respiration rate in thepresence of atractyloside) were similar to control values (Table2). Moreover, the activities of each respiratory chain complexgave normal responses to the different stimuli. This suggeststhat the intrinsic muscle oxidative capacities are normal in LRexcluding complex I and IV inhibitions by anticalcineurins, asalready reported in heart transplant recipients (46). On the

Fig. 7. Schematic representation of the intracellular energetic unit (ICEU). The mitochondria, the sarcoplasmic reticulum, and Mg-ATPase of myofibrils areinterconnected by metabolic channeling of reaction intermediates and energy transfer within the ICEU by the creatine kinase (CK)-PCr and myokinase systems.The protein factors (still unknown and marked as “X”), most probably connected to cytoskeleton, fix the position of mitochondria and probably also control thepermeability of the VDAC channels for ADP and ATP. This increases the microcompartmentation of adenine nucleotides within the ICEU. By interaction withcytoskeletal elements, the mitochondria and sarcoplasmic reticulum are precisely fixed with respect to the structure of the sarcomere of myofibrils between 2z-lines and correspondingly between 2 T tubules. Adenine nucleotides within the ICEU do not equilibrate rapidly with adenine nucleotides in the bulk-waterphase. ATP in the bulk-water phase may constitute a cellular metabolic reserve or serve some regulatory purposes. Adenine nucleotides within the ICEU andbulk-water phase may be connected by some more rapidly diffusing metabolites such as Cr and PCr. AK, adenylate kinase; AKcyt, cytosolic adenylate kinase;CKcyt, cytosolic creatine kinase.

R1151MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

other hand, apparent Km for exogenous ADP was significantlylower in LR compared with C (Fig. 5), mainly explained by thedecreased percentage of type I fibers in LR skeletal muscle(Fig. 2C) (14).

Exercise Training Effects in LR and C

Improvement of apparent Km for exogenous ADP. A sensi-tive parameter resulting from exercise training was the dra-matic improvement in endurance time in LR. To our knowl-edge, it is the second report on benefits of exercise training inLR (34) and the first to use a home-based program for LR. Wehave found significant modifications of the cell energy metab-olism parameters in permeabilized cells. After exercise train-ing, a twofold increase in the apparent Km for exogenous ADPwithout any significant fiber-type transition from glycolytic tooxidative muscles and no alterations in fiber diameter wereobserved in both groups (Figs. 2B and 5 and Table 2). Thisincrease in apparent Km for exogenous ADP resulted in moresignificant effects of creatine on mitochondrial respiratoryparameters. Electron microscopic observations of biopsy ma-terials showed in all cases similar cell structures and intracel-lular arrangement of mitochondria, mostly close to the z-line(Fig. 3). Therefore, the observed changes in biochemical pa-rameters most probably indicated alterations in protein com-plexes in energy transfer pathways as discussed below.

It was found by Ventura-Clapier’s and Bigard’s (18, 44, 45)groups that voluntary activity induced specific alterations in thefast-twitch skeletal muscle, among which was the very signif-icant (several times) increase in the apparent Km for exogenousADP in the regulation of respiration of the skinned cardiacfibers. Our present results give new important information tounderstanding this phenomenon. Our data excluded some plau-sible explanations such as influence of muscle hypertrophy andincrease of the diffusion distances for exogenous ADP (33) andallowed the assumption of exercise-induced alterations in cellstructure and protein complexes.

Creatine/phosphocreatine shuttle and metabolic channeling.In the slow-twitch skeletal muscles, energy is transferredbetween mitochondria and myofibrils by the creatine/phospho-creatine shuttle, whereas in the fast-twitch muscles the energyfor contraction is supplied mostly by the glycolytic system viaequilibrium cytoplasmic creatine kinase and coupled myofi-brillar MM-creatine kinase systems (12, 32, 37, 40). In thepreparations of permeabilized fibers, a stimulating effect ofcreatine on ADP-stimulated respiration indicated a functionalcoupling between the MtCK and ANT by regeneration of localADP (Fig. 1) (32). This local ADP, produced by MtCK in thevicinity of ANT, enters the mitochondrial matrix for rephos-phorylation and activates respiration. These cycles are repeatedmany times, explaining the increase in the rate of respiration atmuch lower exogenous ADP concentrations in the medium inthe presence of creatine, compared with those in its absence.The result is a decrease in apparent Km for exogenous ADP inthe presence of creatine, directly showing the functional cou-pling between MtCK and ANT.

ICEU and Mitochondrial Organization and Interactions

In the fast-twitch skeletal muscle, mitochondria are localizedat the level of I band and close to z-lines and the T-tubularsystem (21, 38). This was mostly the case in the biopsy

samples that we studied (Fig. 3). Oxidative skeletal musclesoccupied intermediate positions between cardiac and glycolyticmuscles with respect to quantity of mitochondria, which werelocalized both at I- and A-band levels (21). The value of theapparent Km for exogenous ADP is very different in oxidativeand glycolytic muscles (14). In the muscle samples with mixedfiber types, the value of the apparent Km for exogenous ADP isbetween two extreme values (300–400 �M and 10–20 �M),depending on the fiber composition (14). These differences thatexist between different types of muscles may be explained bythe different structural and functional organizations of energymetabolism of the cells that exist due to distinct interactionsbetween mitochondria, sarcoplasmic reticulum, and sarco-meres with the cytoskeleton (30). Furthermore, in oxidativecells, the formation of functional complexes between thesestructures, which are called ICEUs (30), gives rise to theunitary nature of energy metabolism in these cells. The struc-ture of these energetic units is described in detail in Fig. 7.

In skeletal muscles with different mitochondrial positions,the structures may be different (30). These complexes play animportant role in metabolic channeling of endogenous ADP byan effective functional coupling between local ADP productionby enzymatic systems that consume ATP and its utilization forATP synthesis in the mitochondrial matrix (Fig. 7) (5). Thesechanneling mechanisms were related to the increased hetero-geneity of the intracellular diffusion of ADP and ATP and localrestrictions of their intracellular diffusion, due to the specificstructural organization of the ICEUs. Thus, according to thislimitation in adenine nucleotide diffusion, the increased valueof the apparent Km for exogenous ADP in permeabilized fibersshowed a higher degree of structural organization of energymetabolism (30).

The role of the structural organization of the cells wasclearly seen in other experiments after a nonselective proteo-lytic treatment of permeabilized fibers from oxidative muscleswith trypsin (1 �M) (13). After this treatment, intracellulararrangement was lost and apparent Km for exogenous ADP wasdecreased close to the value of isolated mitochondria (13).These data most probably show that some or many cytoskeletalproteins could be responsible for this specific cytoarchitectureand also for regulation of mitochondrial function within themuscle cells (6, 19).

Additionally, transgenic animals depleted of cytoskeletalproteins such as desmin, the predominant protein in musclecells, display significant alterations of mitochondrial positionin the cells and regulation of their respiration in situ (13). Theproteins that may control the permeability of the mitochondrialouter membrane within the structure of ICEUs are shown inFig. 7. The nature of the proteins that control mitochondrialposition and function in the cells, however, is still not com-pletely clear.

A complete picture will probably require the integration ofdata obtained from functional genomics approaches and mod-eling efforts. Progress toward this goal will come in the formof high-throughput methods to identifying the connectionswithin the protein-protein and protein-DNA interaction net-works. Thus the recent advances in these areas may provide uswith a first glimpse of the overall structure of molecularinteraction networks in this complex biological system. Theiridentification would be an interesting further task of muscle

R1152 MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

cell proteomic analysis to find an explanation for the interest-ing phenomenon observed in this work.

Exercise activities may induce changes in the expression ofthese proteins and thus increase the restrictions of the exoge-nous ADP diffusion into the cells. The increase in restrictionbarriers may contribute to a better metabolic channeling withinICEUs, making the role of the creatine kinase-phosphocreatinepathway more important in the regulation of respiration. Theobserved statistically significant increase in the Vmax of mito-chondrial respiration measured in the presence of creatine isconsistent with this conclusion and may partially explain theincrease in VO2 max in LR after exercise training (Table 2).These sensitive events could occur at an early stage and do notallow us to make any clear correlation with the integratedphysiological parameters as they concern the intracellular leveland do not involve muscle fiber changes (see Limitations of theStudy). These processes could be compared with the early stageof cardiac ischemia, in which the functional coupling betweenMtCK and ANT is lost while the heart still has functionalproperties (27).

Limitations of the Study

Although two women were included in LR and none in C,this fact did not reach clinical and statistical significance andmay not explain differences between LR and C before and afterexercise training. More importantly, the 10-yr difference infavor of C compared with LR may explain the higher exercisecapacity in C. Actually, as previously reported (24), the mito-chondrial theory of aging does not explain the age-relateddecline of muscle performance in young and elderly healthyadults since mitochondrial oxidative capacity and ATP forma-tion are unchanged.

Before exercise training, highly significant correlations be-tween integrated physiological data and bioenergetic assess-ment at the cellular level were obtained, suggesting a role ofthe ICEUs in physical performance. This kind of correlationbetween improvement in effort tolerance and changes in intra-cellular metabolic channeling (apparent Km for exogenousADP and creatine effect) was not achieved after exercisetraining. This could be related to a type II error. Our hypothesisis that these changes, picked up at the cellular level, occur atthe very early stages. This lack of correlation might be due tothe methods that we used to assess muscular performance. Inthis regard, a segmental endurance muscular assessment wouldhave resulted in better correlations between muscle perfor-mance and bioenergetic studies (26).

Conclusion

Muscle dysfunction in LR is mostly related to pretransplantfactors because anticalcineurins did not result in mitochondrialdysfunction. Before exercise training, in LR and C, exercisecapacity is related to skeletal muscle structure, i.e., fiber typeand ICEU organization. Home exercise training is feasible andefficient in both LR and C. After exercise training, ICEUs exhib-ited a higher control of mitochondrial respiration by creatinelinked to a more efficient functional coupling of ANT-MtCK,resulting in better exercise performances in C and LR.

ACKNOWLEDGMENTS

The Centre d’Investigation Clinique, Inserm of the Centre HopsitalierUniversitaire de Grenoble, and Beatrice Rival and Cecile Cherion (research

assistants), as well as technicians from the pathology laboratory and thepulmonary function test laboratory, are acknowledged for logistic support. Weare indebted to Elisabeth Maclet and technicians of AGIR a Dom, a home careassociation, for the excellent care they provided to the subjects for homeexercise training. We are deeply indebted to our volunteers for enthusiasticparticipation. The contribution of Laurence Kay to some parts of the discussionis also gratefully acknowledged. We thank Dr. Dan Veale (Fellow of the RoyalCollege of Physicians) for reviewing the English text.

GRANTS

This work was supported by grants from Conseil Scientifique of Associa-tion Nationale de Traitement a Domicile Innovation et Recherche, ConseilScientifique of AGIRaDom, Comite departemental de Lutte contre les Mala-dies Respiratoires de l’Isere, Delegation a la Recherche Clinique du CentreHospitalier Universitaire de Grenoble, Centre d’Investigation Clinique, In-serm, CHU Grenoble, and Programme Interdisciplinaire Complexite du Vivantet Action STIC-Inserm. This work was also supported in part by Marie CurieFellowship of the European Community program “Improving Human Re-search Potential and the Socio-economic Knowledge Base” (M. Vendelin,HPMF-CT-2002-01914) and Estonian Science Foundation Grants 5515 and6142.

REFERENCES

1. Acharyya S, Ladner KJ, Nelsen LL, Damrauer J, Reiser PJ, Swoap S,and Guttridge DC. Cancer cachexia is regulated by selective targeting ofskeletal muscle gene products. J Clin Invest 14: 370–378, 2004.

2. Agusti AG, Noguera A, Sauleda J, Sala E, Pons J, and Busquets X.Systemic effects of chronic obstructive pulmonary disease. Eur Respir J21: 347–360, 2003.

3. American Thoracic Society. Skeletal muscle dysfunction in chronicobstructive pulmonary disease. Am J Respir Crit Care Med 159: S1–S40,1999.

4. American Thoracic Society. Pulmonary rehabilitation: 1999. Am J RespirCrit Care Med 159: 1666–1682, 1999.

5. Andrienko T, Kuznetsov AV, Kaambre T, Usson Y, Orosco A, AppaixF, Tiivel T, Sikk P, Vendelin M, Margreiter R, and Saks V. Metabolicconsequences of functional complexes of mitochondria, myofibrils andsarcoplasmic reticulum in muscle cells. J Exp Biol 206: 2059–2072, 2003.

6. Capetanaki Y. Desmin cytoskeleton: a potential regulator of musclemitochondrial behavior and function. Trends Cardiovasc Med 12: 339–348, 2002.

7. Carere R, Patterson GA, Liu P, Williams T, Maurer J, and GrossmanR. Right and left ventricular performance after single and double lungtransplantation: the Toronto Lung Transplant Group. J Thorac CardiovascSurg 102: 115–122, 1991.

8. Dubowitz V and Brookes G. Muscle Biopsy: A Practical Approach (2nded.). London: Bailliere Tindall, 1985.

9. Evans AB, Al-Himyary AJ, Hrovat MI, Pappagianopoulos P, WainJC, Ginns LC, and Systrom DM. Abnormal skeletal muscle oxidativecapacity after lung transplantation by 31P-MRS. Am J Respir Crit CareMed 155: 615–621, 1997.

10. Hall MJ, Snell GI, Side EA, Esmore DS, Walters EH, and WilliamsTJ. Exercise, potassium, and muscle deconditioning postthoracic organtransplantation. J Appl Physiol 77: 2784–2790, 1994.

11. Jakobsson P, Jorfeldt L, and Henriksson J. Metabolic enzyme activityin the quadriceps femoris muscle in patients with severe chronic obstruc-tive pulmonary disease. Am J Respir Crit Care Med 151: 374–377, 1995.

12. Kay L, Nicolay K, Wieringa B, Saks V, and Wallimann T. Directevidence for the control of mitochondrial respiration by mitochondrialcreatine kinase in oxidative muscle cells in situ. J Biol Chem 275:6937–6944, 2000.

13. Kay L, Li Z, Mericskay M, Olivares J, Tranqui L, Fontaine E, TiivelT, Sikk P, Kaambre T, Samuel JL, Rappaport L, Usson Y, Leverve X,Paulin D, and Saks VA. Study of regulation of mitochondrial respirationin vivo. An analysis of influence of ADP diffusion and possible role ofcytoskeleton. Biochim Biophys Acta 1322: 41–59, 1997.

14. Kuznetsov AV, Tiivel T, Sikk P, Kaambre T, Kay L, Daneshrad Z,Rossi A, Kadaja L, Peet N, Seppet E, and Saks VA. Striking differencebetween slow and fast twitch muscles in the kinetics of regulation ofrespiration by ADP in the cells in vivo. Eur J Biochem 241: 909–915,1996.

R1153MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from

15. Kyle UG, Genton L, Karsegard L, Slosman DO, and Pichard C. Singleprediction equation for bioelectrical impedance analysis in adults aged20–94 years. Nutrition 17: 248–253, 2001.

16. Lands LC, Smountas AA, Mesiano G, Brosseau L, Shennib H, Char-bonneau M, and Gauthier R. Maximal exercise capacity and peripheralskeletal muscle function following lung transplantation. J Heart LungTransplant 18: 113–120, 1999.

17. McKenna MJ, Fraser SF, Li JL, Wang XN, Carey MF, Side EA,Morton J, Snell GI, Kjeldsen K, and Williams TJ. Impaired muscleCa2� and K� regulation contribute to poor exercise performance post-lungtransplantation. J Appl Physiol 95: 1606–1616, 2003.

18. Mettauer B, Zoll J, Sanchez H, Lampert E, Ribera F, Veksler V,Bigard X, Mateo P, Epailly E, Lonsdorfer J, and Ventura-Clapier R.Oxidative capacity of skeletal muscle in heart failure patients versussedentary or active control subjects. J Am Coll Cardiol 38: 947–954, 2001.

19. Milner DJ, Mavroidis M, Weisleder N, and Capetanaki Y. Desmincytoskeleton linked to muscle mitochondrial distribution and respiratoryfunction. J Cell Biol 150: 1283–1298, 2000.

20. Miyoshi S, Trulock EP, Schaefers HJ, Hsieh CM, Patterson GA, andCooper JD. Cardiopulmonary exercise testing after single and doublelung transplantation. Chest 97: 1130–1136, 1990.

21. Ogata T and Yamasaki Y. Ultra-high-resolution scanning electron mi-croscopy of mitochondria and sarcoplasmic reticulum arrangement inhuman red, white, and intermediate muscle fibers. Anat Rec 248: 214–223,1997.

22. Pantoja JG, Andrade FH, Stokic DS, Frost AE, Eschenbacher WL,and Reid MB. Respiratory and limb muscle function in lung allograftrecipients. Am J Respir Crit Care Med 160: 1205–1211, 1999.

23. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, andYernault JC. Lung volumes and forced ventilatory flows. Report: Work-ing Party Standardization of Lung Function Tests, European Communityfor Steel and Coal Official Statement of the European Respiratory Society.Eur Respir J 16: 5–40, 1993.

24. Rasmussen UF, Krustrup P, Kjaer M, and Rasmussen HN. Humanskeletal muscle mitochondrial metabolism in youth and senescence: nosigns of functional changes in ATP formation and mitochondrial oxidativecapacity. Pflugers Arch 446: 270–278, 2003.

25. Ribera F, N’Guessan B, Zoll J, Fortin D, Serrurier B, Mettauer B,Bigard X, Ventura-Clapier R, and Lampert E. Mitochondrial electrontransport chain function is enhanced in inspiratory muscles of patients withchronic obstructive pulmonary disease. Am J Respir Crit Care Med 167:873–879, 2003.

26. Richardson RS, Leek BT, Gavin TP, Haseler LG, Mudaliar SRD,Henry R, Mathieu-Costello O, and Wagner PD. Reduced mechanicalefficiency in chronic obstructive pulmonary disease but normal peak VO2

with small muscle mass exercise. Am J Respir Crit Care Med 169: 89–96,2004.

27. Rossi A, Kay L, and Saks V. Early ischemia-induced alterations of theouter mitochondrial membrane and the intermembrane space: a potentialcause for altered energy transfer in cardiac muscle? Mol Cell Biochem184: 401–408, 1998.

28. Sanchez H, Zoll J, Bigard X, Veksler V, Mettauer B, Lampert E,Lonsdorfer J, and Ventura-Clapier R. Effect of cyclosporin A and itsvehicle on cardiac and skeletal muscle mitochondria: relationship toefficacy of the respiratory chain. Br J Pharmacol 133: 781–788, 2001.

29. Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, TranquiL, Olivares J, Winkler K, Wiedemann F, and Kunz WS. Permeabilizedcell and skinned fiber techniques in studies of mitochondrial function invivo. Mol Cell Biochem 184: 81–100, 1998.

30. Saks V, Kaambre T, Sikk P, Eimre M, Orlova E, Paju K, Piirsoo A,Appaix F, Kay L, Regiz-Zagrosek V, Fleck E, and Seppet E. Intracel-lular energetic units in red muscle cells. Biochem J 356: 643–657, 2001.

31. Saks V, Kuznetsov A, Andrienko T, Usson Y, Appaix F, Guerrero K,Kaambre T, Sikk P, Lemba M, and Vendelin M. Heterogeneity of ADPdiffusion and regulation of respiration in cardiac cells. Biophys J 84:3436–3456, 2003.

32. Saks VA, Kuznetsov AV, Vendelin M, Guerrero K, Kay L, and SeppetEK. Functional coupling as a basic mechanism of feedback regulation ofcardiac energy metabolism. Mol Cell Biochem 256–257: 185–199, 2004.

33. Seppet EK, Eimre M, Andrienko T, Kaambre T, Sikk P, KuznetsovAV, and Saks V. Studies of mitochondrial respiration in muscle cells insitu: use and misuse of experimental evidence in mathematical modelling.Mol Cell Biochem 256–257: 219–227, 2004.

34. Stiebellehner L, Quittan M, End A, Wieselthaler G, Klepetko W,Haber P, and Burghuber OC. Aerobic endurance training programimproves exercise performance in lung transplant recipients. Chest 113:906–912, 1998.

35. Trulock EP. Lung transplantation. Am J Respir Crit Care Med 155:789–818, 1997.

36. Vendelin M, Eimre M, Seppet E, Peet N, Andrienko T, Lemba M,Engelbrecht J, Seppet EK, and Saks VA. Intracellular diffusion ofadenosine phosphates is locally restricted in cardiac muscle. Mol CellBiochem 256–257: 229–241, 2004.

37. Vendelin M, Lemba M, and Saks VA. Analysis of functional coupling:mitochondrial creatine kinase and adenine nucleotide translocase. BiophysJ 87: 696–713, 2004.

38. Vendelin M, Beraud N, Guerrero K, Andrienko T, Kuznetsov AV,Olivares J, Kay L, and Saks VA. Mitochondrial regular arrangement inmuscle cells: a “crystal-like” pattern. Am J Physiol Cell Physiol 288:C757–C767, 2005.

39. Ventura-Clapier R, Kaasik A, and Veksler V. Structural and functionaladaptations of striated muscles to CK deficiency. Mol Cell Biochem256–257: 29–41, 2004.

40. Walsh B, Tonkonogi M, Soderlund K, Hultman E, Saks V, and SahlinK. The role of phosphorylcreatine and creatine in the regulation ofmitochondrial respiration in human skeletal muscle. J Physiol 537: 971–978, 2001.

41. Wang XN, Williams TJ, McKenna MJ, Li JL, Fraser SF, Side EA,Snell GI, Walters EH, and Carey MF. Skeletal muscle oxidativecapacity, fiber type, and metabolites after lung transplantation. Am JRespir Crit Care Med 160: 57–63, 1999.

42. Williams TJ, Patterson GA, McClean PA, Zamel N, and Maurer JR.Maximal exercise testing in single and double lung transplant recipients.Am Rev Respir Dis 145: 101–105, 1992.

43. William RS, Morton J, and Williams TJ. Exercise after lung transplant.Curr Opin Organ Transpl 7: 265–270, 2002.

44. Zoll J, Koulmann N, Bahi L, Ventura-Clapier R, and Bigard AX.Quantitative and qualitative adaptation of skeletal muscle mitochondria toincreased physical activity. J Cell Physiol 194: 186–193, 2002.

45. Zoll J, Sanchez H, N’Guessan B, Ribera F, Lampert E, Bigard X,Serrurier B, Fortin D, Geny B, Veksler V, Ventura-Clapier R, andMettauer B. Physical activity changes the regulation of mitochondrialrespiration in human skeletal muscle. J Physiol 543: 191–200, 2002.

46. Zoll J, N’Guessan B, Ribera F, Lampert E, Fortin D, Veksler V,Bigard X, Geny B, Lonsdorfer J, Ventura-Clapier R, and Mettauer B.Preserved response of mitochondrial function to short-term endurancetraining in skeletal muscle of heart transplant recipients. J Am Coll Cardiol42: 126–132, 2003.

R1154 MUSCLE BIOENERGETICS IN LUNG TRANSPLANTATION

AJP-Regul Integr Comp Physiol • VOL 289 • OCTOBER 2005 • www.ajpregu.org

on Septem

ber 24, 2005 ajpregu.physiology.org

Dow

nloaded from